Incidence and Transmission Dynamics of Bordetella pertussis Infection in Rural and Urban Communities, South Africa, 2016‒2018

We conducted 3 prospective cohort studies (2016–2018), enrolling persons from 2 communities in South Africa. Nasopharyngeal swab specimens were collected twice a week from participants. Factors associated with Bordetella pertussis incidence, episode duration, and household transmission were determined by using Poisson regression, Weibull accelerated time-failure, and logistic regression hierarchical models, respectively. Among 1,684 participants, 118 episodes of infection were detected in 107 participants (incidence 0.21, 95% CI 0.17–0.25 infections/100 person-weeks). Children <5 years of age who had incomplete vaccination were more likely to have pertussis infection. Episode duration was longer for participants who had higher bacterial loads. Transmission was more likely to occur from male index case-patients and persons who had >7 days infection duration. In both communities, there was high incidence of B. pertussis infection and most cases were colonized.

We conducted 3 prospective cohort studies (2016-2018), enrolling persons from 2 communities in South Africa. Nasopharyngeal swab specimens were collected twice a week from participants. Factors associated with Bordetella pertussis incidence, episode duration, and household transmission were determined by using Poisson regression, Weibull accelerated time-failure, and logistic regression hierarchical models, respectively. Among 1,684 participants, 118 episodes of infection were detected in 107 participants (incidence 0.21, 95% CI 0.17-0.25 infections/100 person-weeks). Children <5 years of age who had incomplete vaccination were more likely to have pertussis infection. Episode duration was longer for participants who had higher bacterial loads. Transmission was more likely to occur from male index case-patients and persons who had >7 days infection duration. In both communities, there was high incidence of B. pertussis infection and most cases were colonized.
2013-2018, among persons hospitalized because of pneumonia, annual B. pertussis incidence was 17 cases/100,000 population, and the highest incidence of disease and most deaths were in children <1 year of age (9). In 2018, clusters of pertussis were observed in several provinces in South Africa, and 54% of cases were detected in infants <3 months of age (10). Infants <1 year of age were at highest risk for severe pertussis and death. It is essential to understand infection in older children and adults because these age groups are likely sources of transmission to infants (3,5). This study reports the incidence, factors associated with infection, duration of infection, and transmission dynamics in persons of all ages in an urban community and a rural community in South Africa.

Study Population
During 2016-2018 (May-October 2016, January-October 2017, and January-October 2018), we conducted a community cohort study in 2 communities in South Africa: 1 rural (Agincourt, Mpumalanga Province) and 1 urban (Klerksdorp, North West Province). We enrolled consenting members of randomly selected households each year. During the household enrollment process, at least 80% of the household members within each household had to consent to be included in the study for a household to be enrolled. A detailed account of the study methods and the cohort profile has been previously reported (11,12).

Household Visits
Demographic and baseline health status (including HIV) were collected for all participants at enrollment. Participants were considered HIV infected if they had 1 of the following during the follow-up period: 2 positive rapid HIV test results, evidence of a positive HIV laboratory result, or evidence of receiving antiretroviral treatment. Vaccination status was obtained for children <5 years of age from Road-to-Health Cards, which serve as a child's formal health record in South Africa. Households were visited twice a week for nasopharyngeal specimen collection and symptom determination by using a structured questionnaire. Symptom data included fever (self-reported or measured tympanic temperature >38°C), cough, difficulty breathing, sore throat, nasal congestion, chest pain, muscle aches, headache, vomiting, or diarrhea. In addition, during 2018, persons who tested PCR positive for B. pertussis were retrospectively interviewed (interviews were conducted immediately after a positive PCR result) to confirm if the persons experienced any pertussisspecific symptoms (cough, inspiratory whoop, posttussive vomiting, or apnea) during the infection.

Specimen Collection and Testing
At each visit, a nasopharyngeal swab specimen was collected and placed in PrimeStore Molecular Transport Medium (Longhorn Vaccines & Diagnostics, https://lhnvd.com) and transported to the laboratory within 48-72 hours for testing. Specimens were received in the laboratory in real-time and then batched for further processing. Total nucleic acids were extracted by using the Roche MagNA Pure 96 Instrument (Roche Diagnostics, https://www. roche.com) and the MP96 DNA and Viral NA SV Kits (Roche Diagnostics). Extracts were tested by using an internally validated IS481 and human ribonuclease P (RNaseP) duplex real-time PCR. Any specimen that tested positive for IS481 with a cycle threshold (Ct) value <45 was repeated (from extraction using a fresh aliquot) and retested (2 replicates) by using a second, multitarget real-time PCR targeting B. pertussis, B. parapertussis, and B. holmesii (13). A specimen was considered positive for B. pertussis if IS481 or ptxS1 targets was detected with Ct values <45 in at least 2/3 replicates and negative for hIS1001 (B. holmesii) and pIS1001 (B. parapertussis).

Study Definitions and Data Analysis
We defined an episode of B. pertussis infection as >1 consecutive visits in which B. pertussis was detected. A new episode was one that began after a period of at least 12 consecutive visits (i.e., 6 weeks) in which B. pertussis was not detected. We determined the incidence of B. pertussis infection by dividing the number of PCR-positive episodes by the person-time under observation, expressed as 100 person-weeks. We assessed factors associated with incidence by using Poisson regression accounting for the person-time under observation. For analysis of incidence, we considered all identified episodes of infections, including multiple episodes in the same person.
We estimated the duration of a B. pertussis infection as the date of the last positive specimen minus the date of the first positive specimen (within the same episode of infection) plus 2.5 days to account for gaps between visits; values are expressed as mean +SD. We performed analysis of factors associated with the duration of infection (time-to-event outcome) by using accelerated time failure Weibull regression and analysis of B. pertussis bacterial loads by using IS481 Ct as a proxy for bacterial load. We characterized IS481 bacterial load as follows: Ct <34, higher bacterial load; Ct 34-39, intermediate bacterial load; Ct 40-44, lower bacterial load.
We defined an episode of symptomatic B. pertussis infection as illness in a person who had >1 symptoms reported from 1 visit before to 1 visit after the B. pertussis episode. Signs and symptoms included fever, cough, difficulty breathing, sore throat, nasal congestion, chest pain, muscle aches, headache, vomiting, and diarrhea. In 2018, we used the pertussisspecific symptom data collected to determine if positive cases could be classified as pertussis by using the South Africa notifiable medical condition pertussis case definition (cough lasting >14 days, or cough of any duration for children <1 year of age, along with >1 of the following symptoms: paroxysms of coughing, inspiratory whoop, posttussive vomiting, and apnea [with or without cyanosis; only for infants <1 year of age]).
A cluster of cases included all B. pertussis infections in a single household that occurred within an interval of <7 days. The index case was the first person testing positive within a cluster. We estimated cluster duration as the time from the first day of positivity of the first person in a cluster to the last day of positivity of the last person. We defined household cumulative infection risk (HCIR) as the proportion of subsequent infections within a household after the first PCR-positive case and evaluated HCIR among all households in which >1 household member had B. pertussis infection. The formula for HCIR was the number of infected persons within a household (excluding the index case) divided by number of enrolled persons living within the household. We excluded households that had coprimary cases from the HCIR analysis. We defined crowding as >2 persons sharing a sleeping room within the household.
We used logistic regression to analyze factors associated with symptomatic fraction and HCIR. Pertussis vaccine status was based on number of vaccine doses received by age. For all analyses, we accounted for clustering by study site and households within site by using hierarchical mixed effect models. For the multivariable models, we assessed all variables that were significant at p<0.2 by univariate analysis and removed nonsignificant factors (p>0.05) by using manual backward elimination. We set statistical significance at p<0.05 and performed all statistical analyses in Stata version 14.1 (StataCorp LP, https:// www.stata.com).

Ethics
Ethics approval for the Prospective Household cohort study of Influenza, Respiratory Syncytial virus, and other respiratory pathogens community burden and Transmission dynamics in South Africa (PHIRST) was obtained from the University of the Witwatersrand Human Research Ethics Committee, South Africa (protocol no. 150808, C.C.). Written informed consent was obtained from all enrolled persons, or parents/guardians in the case of minors. A separate ethics application for the pertussis component of the PHIRST was obtained from the University of the Witwatersrand Human Research Ethics Committee, South Africa (protocol no. M210676, F.M.).

Incidence and Factors Associated with Incidence
Among 1,684 study participants, 118 episodes of B. pertussis infection were detected in 107 participants; 11 persons had 2 episodes of B. pertussis infection. There were no demographic/clinical differences observed between persons who had 1 or 2 episodes of infection. The incidence of B. pertussis infection was 0.21 (95% CI 0.17-0.25)/100 person-weeks (Table 2). The highest incidence of B. pertussis infection was observed in persons 5-14 years of age (0.27 [95% CI 0.20-0.35]/100 person-weeks). Children <5 years of age who had incomplete vaccination for age were more likely to be infected with B. pertussis than were vaccinated children (adjusted incidence rate ratio 4.48, 95% CI 1.38-14.59). Among the 36 enrolled infants, 5 B. pertussis infections were identified in those 9-11 months of age. Of those infants, 4 were fully vaccinated for age; 1 infant (incomplete vaccination) had symptoms (runny nose) during the B. pertussis infection.

Duration of PCR-Positive B. pertussis Infection
The mean ± SD duration of B. pertussis infection was 12.0 +19.1 days. The duration of B. pertussis infection was longer when >2 symptoms were reported (adjusted hazard ratio 0.26, 95% CI 0.18-0.67) (

Discussion
In this study population, we found a high incidence of B. pertussis and most infected persons were colonized. We detected 118 episodes of B. pertussis infection in 107 participants, of which 11 persons had 2 episodes. The overall HCIR was 14%, and 39% of index cases in the household were colonized with B. pertussis. The mean duration of PCR-positive B. pertussis infection was 12 days. Persons positive for B. pertussis were more likely to report symptoms if they were infected for >7 days. Transmission was more likely to occur from male index case-patients and persons who had longer episode duration. In this community cohort study of healthy persons in South Africa, the incidence of B. pertussis was 0.21 cases/100 person-weeks. Incidence in this study was higher than the mean annual incidence risk of 17 cases/100,000 population previously reported in South Africa for persons hospitalized because of pneumonia during 2013-2018 (9). This difference was probably caused by the fact that the study conducted by Wolter et al. (9) was a cross-sectional study enrolling hospitalized patients who had respiratory illness, whereas our study was a community cohort study enrolling healthy persons. It is difficult to compare our incidence with those of other studies that report incidence data because our study was a community cohort study, whereas other studies focus on populations with B. pertussis disease or populations that had outbreaks of B. pertussis. Thus, more data describing the incidence of B. pertussis among healthy persons over time, and additional longitudinal community data are required for meaningful comparisons to be made. Assessing factors associated with B. pertussis incidence, children <5 years of age who did not receive the full schedule of pertussis vaccine for age were more likely to be infected with B. pertussis than were vaccinated children. Similarly, other studies in South Africa showed that risk for pertussis and risk for hospitalization caused by pertussis decreased when persons were vaccinated (9,14). Data on duration of PCR-positive B. pertussis infection among persons are limited. In our study, the average duration of naturally acquired B. pertussis infection was 12 days and episode duration was longer for participants with higher bacterial loads, similar to findings for influenza virus infection in the same cohort (11). A human challenge study that induced B. pertussis colonization in healthy adults showed that B. pertussis persisted within the nasopharynx for up to 16 days postinoculation in some of the study participants (15). However, it should be noted that in the human challenge study, adults had received vaccination within the previous 5 years and were treated for colonization from day 14. In our study, data on vaccination of persons >5 years of age and treatment were missing, and that information could have potentially affected the duration of B. pertussis within the nasopharynx of study participants. Symptoms associated with B. pertussis PCRpositive cases vary from mild, nonspecific respiratory symptoms to pertussis-specific symptoms. In this study, symptomatic B. pertussis-infected persons reported nonspecific respiratory symptoms, such as runny nose, cough, fever, or sore throat. For the 2018 subset of symptomatic persons who were investigated for B. pertussis-specific symptoms, 60% met the notifiable medical condition pertussis case definition. A study in Spain determining factors influencing the spread of B. pertussis in households found that participants of all ages experienced B. pertussis-specific signs and symptoms, such as cough lasting >2 weeks, paroxysmal cough, posttussive vomiting, inspiratory stridor, apnea, and fever (16). In South Africa, common signs and symptoms reported in children hospitalized with laboratory pertussis were nonspecific cough and fever (17). Evidence suggests that colonization is a major trait of the B. pertussis pathogen. In our study, two thirds of persons infected with B. pertussis did not report any symptoms. B. pertussis incidence data from the United States and the United Kingdom provided evidence of colonization of B. pertussis (18). de Graaf et al. showed that colonization could be induced by the intranasal inoculation of B. pertussis in a human challenge model (15). Warfel et al. found that persons previously vaccinated with the acellular pertussis vaccine can become colonized with B. pertussis (19). There is evidence in the literature supporting colonization of B. pertussis in the nasopharynx of persons, which was also observed in our study. In addition to colonization, we found a high proportion of index case-patients transmitting B. pertussis to other household contacts. However, it is difficult to draw any conclusions from that result because additional longitudinal community data are required for meaningful comparisons to be made. The transmission dynamics of B. pertussis within households is poorly understood and requires additional research. Within the PHIRST, the overall HCIR was 14%. In 2001, a pertussis outbreak in New South Wales in Australia showed a 22.3% secondary household attack rate (20) and increased risk for household transmission when the index case-patient began antimicrobial drug treatment >7 days after onset of symptoms (20). Transmission in our study was associated with male sex, and the index case-patient had a long episode duration (>7 days). The association between prolonged episode duration and increased transmission is biologically plausible and has been demonstrated for other pathogens, such as influenza virus (11). Previous studies have not documented a difference in transmission between male and female patients (15,20), so this finding should be further explored.
The first limitation of our study is that it was conducted at only 2 sites in South Africa, and results might not be generalizable to other areas and communities within this country. Over the study period, specimens were collected from each person twice a week for a period of 6-10 months. Because we did not collect samples for a full year and therefore could not accurately estimate the incidence over the period of a year, we presented incidence as per 100 personweeks. Vaccination status was confirmed only for children <5 years of age, so we could conduct analyses regarding vaccination only for this age group. Symptom data collection was poor for 2016 but improved in 2017 and 2018; therefore, the symptom data presented could be an underestimate. The number of B. pertussis cases detected in this study is small, so some statistical analyses were underpowered.
The PHIRST was unique in that it was a community cohort study investigating B. pertussis infection among healthy persons. A major strength of the study was the repeated, longitudinal sampling of persons over time according to a specified schedule, irrespective of symptoms. This study method ensured that the data collected over time were robust against sampling bias and enabled us to identify the large proportion of persons who were colonized with B. pertussis.
In conclusion, in 2 disparate communities in South Africa, we found a high incidence of B. pertussis, and two thirds of cases were colonizations. We have demonstrated that persons who are colonized with B. pertussis can transmit this bacteria to other household members and thus represent a source of transmission to susceptible age groups.